Transcript
Baedah Madjid DEPART. OF MICROBIOLOGY, MEDICAL
FACULTY, HASANUDIN UNIVERSITY2007
The student must:The student must:
• know what methods are used to determine the know what methods are used to determine the
microbial cause of the diseasemicrobial cause of the disease
• understand how these methods are combines with understand how these methods are combines with
clinical examination and other investigations in clinical examination and other investigations in
clinical practice.clinical practice.
• recognize the limit of laboratory-base evidence in recognize the limit of laboratory-base evidence in
making a specific microbiological diagnosis.making a specific microbiological diagnosis.
DEFENITIVE MICROBIOLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
RATIONAL THERAPY
CONTROL
DEFENITIVEDIAGNOSIS OF INFECTIOUS DISEASES
CLINICAL FEATURES RADIOGRAPH
LABORATORY DIAGNOSIS
PATHOLOGY MICROBIOLOGY CLINICAL CHEMISTRY
ACCURATE MICROBIOLOGY DIAGNOSIS
SPECIMENS
METHODS
INSTRUMENTS/REAGENTS
HUMAN RESOURCES
METHODS :METHODS : → high sensitivity & high → high sensitivity & high specificityspecificity- High sensitivity & High specificity :High sensitivity & High specificity :
- - High sensitivity & Low specificity:High sensitivity & Low specificity:
all infected individuals detected, but no false-all infected individuals detected, but no false-positive responsespositive responses
all infected individuals detected, but many false-all infected individuals detected, but many false-positives response.positives response.
- - Low sensitivity & High specificity:Low sensitivity & High specificity:not all infected individuals detected (some false-not all infected individuals detected (some false-negatives) , but no false-positivesnegatives) , but no false-positives
- - Low sensitivity & Low specificity:Low sensitivity & Low specificity:not all infected individuals are detected (some false-not all infected individuals are detected (some false-negatives) and some who response are falsely positive.negatives) and some who response are falsely positive.
INSTRUMENTS:INSTRUMENTS: valid valid
Depend on:
REAGENTS:REAGENTS: fresh made fresh made
- Expert in his job- Expert in his job - High dedicated- High dedicated
HUMAN RESOURCESHUMAN RESOURCES
SPECIEMENS:SPECIEMENS: appropriate appropriateSputum, faces, blood, etcSputum, faces, blood, etc
Specimen should appropriatelySpecimen should appropriately- CollectedCollected- StoredStored- TransportedTransported- ProcessedProcessed
Specimen amount:Specimen amount: sufficient for the test sufficient for the test
Collecting MethodCollecting Method: Sterile: Sterile
Time of specimen collection:Time of specimen collection: punctual punctual
- Before antimicrobial therapy - Before antimicrobial therapy - Stage of the course of the diseaseStage of the course of the disease- Any time, or early morning, or 24 hours Any time, or early morning, or 24 hours
Location:Location: the site of suspected infection the site of suspected infection
Specimen container:Specimen container: sterile & screw cap sterile & screw cap
Time:Time:
●●Specimen should be processed not more Specimen should be processed not more than 8 hr.than 8 hr.● ● Specimen (in transport medium) Specimen (in transport medium) should be processed in 24 hr.should be processed in 24 hr.
Temperature :emperature : depend on bacteria depend on bacteria suspected: 4suspected: 4ooC, or in room temperatureC, or in room temperature
Specimen should be transported to Specimen should be transported to Laboratory as soon after collection, in:Laboratory as soon after collection, in:
- Transport medium- Transport medium- Room temperature or ice- Room temperature or ice
SPECIMEN
BACTERIA MACROMOLECULE
Bottle/med.transport
DirectPreparation
Isolation & IdentificationMicroscopy
Ag AbDNA OR
RNA
SPECIMEN
DIRECT SMEAR
WET MOUNT STAINING
FLUORESCENCE ACID FASTG R A M
Light microscopy:1. Wet mount: - morphology - motility 2. Preparation a. Gram stain: morphology, Gram positive or negative b. Acid fast stain: morphology, Acid fast bacilli c. Fluorescence: Increasing sensitivity of Acid fast
preparation.
SPECIMEN
ENRICHMENT MEDIUM
BOTTLE OR TRANSPORT MEDIUM
SELECTIVE/DIFFERENTIAL MEDIUM
PURE CULTURE
Identification GRAM BIOCHEM TESTS DNA Tes RAG
1. Transport medium = carrier medium2. Basic medium 3. Enriched medium4. Enrichment medium5. Selective medium6. Differential medium7. Special medium8. Anaerobic medium
1. Colony appearance 2. Gram: Morphology & Gram characteristic3. Motality : solid medium or semi-solid4. Biochemical Reaction5. Macromolecule identification : - Ag microbial detection - Ab detection - DNA/RNA detection
Isolated colony → colony appearance
1. Form and margin
2. Sizes
3. Elevation
4. Clarity
5. Color : pigment, chemical reaction
6. Hemolysis : α, ß, & γ
Colony’s color: Red Colorless
Colony’s color : pigment
Non-hemolysisß - Hemolysis
1. Charbohydrate Metabolism
- Test for sugar fermentation, VP-MR Test
2. Protein Metabolism
- Indol test
3. Lipid Metabolism ( rare)
5. Single enzyme test
Catalase Test, coagulase test, oxidase test, urease test
glucose lactose
Triple Sugar Iron Agar
Citrat Medium
DNAse Medium
Invitro Ag-Ab reacton:1. Agglutination Test2. Precipitation & flocculation Test3. Complement fixation Test4. Immuno-fluorescent5. Radio-immuno assay (RIA)6. Enzyme linked immunosoebent assay
(ELISA)7. Blotting immuno assay
1. Direct agglutination :
- Widal
- Gol. Darah
2. Particle agglutination
a. Latex Agglutination Test
b. Co-agglutination Test
c. Hemagglutination (TPHA)
A. Flocculation Test
- VDRL
- RPR
B. Precipitation Test
1. Immune-double diffusion : Ag orAb.
detection
2. Radial immunodiffusion: qualitative &
quantitative.
1. Solid phase ELISA
a. Direct
b. Indirect
Qualitative & quantitative
2. Membrane bound SPIA
Qualitative
1. Gen-Probe (Hybridization)
- Less sensitive
2. Polymerase chain reaction (PCR)
- High sensitivity
● RFLP (Retriction Fragment Length
Polymorphism): polymorphism
● DNA Sequencing
A.Diffusion Test:
● Disc Diffusion test
B. Dilution test:
● Tube Dilution test
● Microbroth Panel Dilution Test
C. Combine : E. Susceptibility Test
Disc Diffusion test:
- R or S“E. Test susceptibility”
MBC & MIC
• MBC: Minimal Bactericidal Concentration
• MIC: Minimal Inhibitory Concentration
Microbroth Panel Test
MBC & MIC
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